The Fas ligand (FasL, also CD95L or Apo-1L) belongs to the tumor necrosis factor (TNF) family. It associates with the Fas receptor (Fas, also CD95 or Apo-1). Both function primarily as membrane-bound cell-surface proteins. The interaction between Fas and FasL is a key regulator of apoptosis within the immune system. Binding of FasL by Fas triggers apoptosis. Since both Fas and FasL are typically membrane-bound, cells expressing either Fas or FasL generally must come into contact with cells expressing the other in order to induce cell death (Rowe, P. M., Lancet, 1996, 347, 1398). Under normal conditions, expression of the FasL is generally limited to activated T cells and macrophages. Fas is expressed in a variety of lymphoid and non-lymphoid cells including thymus, liver, heart and kidney (Watanabe-Fukunaga, R., et al., J. Immunol., 1992, 148, 1274-1279).
Expression of FasL is involved in a number of cancers, including lymphomas, melanoma (Hahne, M., et al., Science, 1996, 274, 1363-1366), colon, hepatic and lung carcinomas and astrocytomas (Saas, P., et al., J. Clin. Invest., 1997, 99, 1173-1178). It is thought that FasL expression by tumor cells is a mechanism by which they escape killing by the immune system and instead enables them to kill immune cells possessing Fas receptor on their surfaces (Walker, P. R., et al., J. Immunol., 1997, 158, 4521-4524).
Fas and FasL are also involved in other diseases, including autoimmune and inflammatory diseases. These include Hashimoto's thyroiditis (Giordano, C., et al., Science, 1997, 275, 1189-1192), hepatitis (Kondo, T., et al., Nat. Med., 1997, 3, 409-413), diabetes (Chervonsky, A. V., et al., Cell, 1997, 89, 17-24), myasthenia gravis (Moulian, N., et al., Blood, 1997, 89, 3287-3295), ulcerative colitis (Strater, J., et al., Gastroenterology, 1997, 113, 160-167), autoimmune gastritis (Nishio, A., et al., Gastroenterology 1996, 111, 959-967), Sjogren's syndrome (Kong, L., et al., Arthritis Rheum., 1997, 40, 87-97) and HIV infection (Sieg, S., et al., Proc. Natl. Acad. Sci (USA), 1997, 94, 5860-5865).
Fap-1 (Fas associated protein 1 or protein tyrosine phosphatase (PTP-BAS, type 1)) is a tyrosine phosphatase that binds with a negative regulatory element of Fas (Sato, T., et al., Science, 1995, 268, 411-415). It also is an inhibitor of Fas-mediated apoptosis and an important component of Fas mediated signaling. The presence of Fap-1 in tumor cell lines also correlated with resistance to Fas antibody. Takahashi, M. et al. (Gan To Kagaku Ryoho, 1997, 24, 222-228) found that Fap-1 was expressed in many colon cancer cell lines, but not in normal colon cells.
Several approaches have been used to study the interaction between Fas and FasL and could potentially be used for therapeutic purposes. One way to disrupt the balance (altered or normal) between Fas and FasL is to provide additional amounts of one of them. This approach has been used with soluble Fas by Kondo, T., et al. (Nature Med., 1997, 3, 409-413) to prevent hepatitis in a transgenic mouse model and Cheng, J., et al. (Science, 1994, 263, 1759-1762) to inhibit Fas-mediated apoptosis in systemic lupus erythematosus. Arai, H., et al. (Proc. Natl. Acad. Sci. USA, 1997, 94, 13862-13867) used a somewhat different approach to increase FasL. An adenoviral expression vector containing FasL was used to infect tumor cells. The increased levels of FasL induced apoptosis and caused tumor regression.
Portions of these proteins could also be used. It was found that the three C-terminal amino acids of Fas were necessary and sufficient for binding to Fap-1 (Yanagisawa, J., et al., J. Biol. Chem., 1997, 272, 8539-8545). Introduction of this peptide into a colon cancer cell line induced Fas-mediated apoptosis.
Monoclonal antibodies to Fas have been used extensively to induce apoptosis. Anti-Fas antibodies resulted in tumor regression in B cell tumors (Trauth B. C., et al., Science, 1989, 245, 301-305), adult T-cell leukemia (Debatin, K. M., et al., Lancet, 1990, 335, 497-500), gliomas (Weller, M., et al., J. Clin. Invest., 1994, 94, 954-964), and colorectal cancer (Meterissian, S. H., Ann. Surg. Oncol., 1997, 4, 169-175). Antibodies to Fas also killed HIV infected cells (Kobayashi, N., et al., Proc. Natl. Acad. Sci USA, 1990, 87, 9620-9624). Monoclonal antibodies have been used in combination with chemotherapeutic drugs to overcome drug resistance (Morimoto, H., et al., Cancer Res., 1993, 53, 2591-2596), Nakamura, S., et al., Anticancer Res., 1997, 17, 173-179) and Wakahara, Y., et al., Oncology, 1997, 54, 48-54).
Chemical agents have been used to inhibit FasL expression (Yang, Y., et al., J. Exp. Med., 1995, 181, 1673-1682). Retinoic acid and corticosteroids inhibit the up-regulation of FasL.
An antisense RNA approach, involving the antisense expression of a significant portion of a gene, has been used to modulate expression of Fas and Fap-1. Herr, I. et al. (EMBO J., 1997, 16, 6200-6208) expressed a 360 bp fragment of Fas in the antisense orientation to inhibit apoptosis. Freiss, G. et al. (Mol. Endocrinol., 1998, 12, 568-579) expressed a greater than 600 bp fragment of Fap-1 to inhibit Fap-1 expression.
Oligonucleotides have also been used to modulate expression of FasL. A bifunctional ribozyme targeted to both perforin and FasL was designed to treat graft-versus-host disease (Du, Z., et al., Biochem. Biophys. Res. Commun., 1996 226, 595-600). Antisense oligonucleotides have been used against both Fas and FasL. Yu, W. et al. (Cancer Res., 1999, 59, 953-961) used an oligonucleotide targeted to the translation initiation site of human Fas to reduce Fas mediated signaling in breast cancer cells. Lee, J., et al. (Endocrinology, 1997, 138, 2081-2088) used an oligonucleotide targeted to the translation initiation region of rat FasL to show that Fas system regulates spermatogenesis. Turley, J. M., et al. (Cancer Res., 1997, 57, 881-890) used an oligonucleotide targeted to the translation initiation region of human FasL to show that the Fas system was involved in Vitamin E succinate mediated apoptosis of human breast cancer cells. O'Connell, J., et al. (J. Exp. Med., 1996, 184, 1075-1082) used a model involving Jurkat T cells and SW620, a colon cancer cell line. The presence of FasL on SW620 causes apoptosis of Jurkat cells which possess the Fas receptor. Antisense oligonucleotides to either the FasL on SW620 or Fas on Jurkat cells could prevent apoptosis of the Jurkat cells. Oligonucleotides were designed to target sequences toward the 3' end of the coding region.
There remains a long-felt need for improved compositions and methods for inhibiting Fas, FasL and Fap-1 gene expression.